Tumor Tissue Protein Extraction and Analysis

To produce the lysate from tumor tissue, 100 mg tissue was lysed and homogenized in radio immuno-precipitation Assay (RIPA) buffer (Sigma, Australia) containing 10% protease inhibitor cocktail. The samples were then centrifuged at 10,000 g at 4° C for 10 minutes and the protein content in supernatant was quantified [17 (link)]. Fifty micrograms protein were fixed on 12% gels and electrophoresed for 2 hours at 85 V and transferred to Poly vinylidene Fluoride (PVDF) membranes. The membranes were incubated with the primary antibodies ( VEGF and SPARC, 1:200 dilutions, Santa Cruz Biotechnology) for overnight at 4°C followed by one hour incubation with secondary antibodies (anti-mouse from Santa Cruz Biotechnology and anti-rabbit from Cell Signalling, USA). The bands were visualized by an enhanced chemiluminescence detection kit (GE Healthcare, Australia). The blots were then stripped using Seppro western blot stripping buffer (Sigma, Australia) and re-probed with GAPDH (1:1000 dilution, Sigma, Australia).